This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is the small (xcex3) subunit of human transcription factor IIA, sometimes hereinafter referred to as xe2x80x9csmall subunitxe2x80x9d. The invention also relates to inhibiting the action of such polypeptides.
In prokaryotes, simply mixing purified RNA polymerase, a template carrying a promoter, nucleoside triphosphates, and appropriate buffer and salts is sufficient to obtain specific gene transcription in vitro beginning at the correct sites. Purified RNA polymerase from eukaryotes, however, initiates transcription very poorly and essentially at random. Accordingly, accessory factors are required for accurate initiation of transcription in eukaryotes. Some of these transcription factors are general factors required for initiation at all promoters, while others are gene-specific and are required only for certain promoters. Among the general factors is a protein called Transcription Factor IID xe2x80x9cTFIIDxe2x80x9d, which binds to a TATA sequence, wherein T represents thymidine and A represents adenosine, in promoters. Other general factors are also involved in the assembly of a multicomponent protein complex at the promoter.
In general, transcription factors are found to contain two functional domains, one for DNA-binding and one for transcriptional activation. These functions often reside within circumscribed structural domains that retain their function when removed from their natural context. The DNA-binding domains of transcription factors fall into several structural families based on their primary amino acid sequence.
In order to identify the specific nucleotides that control gene expression, regions of the gene flanking the coding region can be sequenced. Comparisons of these sequences reveal common patterns near the 5xe2x80x2 and 3xe2x80x2 ends of different genes. These are predicted to be important for proper transcription by RNA polymerase. The most common motif is the TATA sequence around 30 bp from the transcriptional start site. Other conserved sequences have been found roughly 50 to 100 bp upstream of the transcriptional start site.
Eukaryotic transcriptional activation requires the characterization of several multiprotein complexes, referred as general transcription factors and coactivators1,2. The heteromeric general transcription factor TFIIA binds directly to the TATA binding protein (TBP)3,4 and has been implicated in the process of transcriptional activation5-8. The xcex3 subunit of TFIIA binds weakly to the TATA binding protein, but strongly stabilized the binding of the large subunit of TFIIA (xcex1 xcex2) to TBP. Recombinant human TFIIA is functional for the transcriptional activation mediated by at least three distinct activators. Both the xcex1 xcex2 and xcex3 subunits are essential for activator dependent stimulation of TFIID by binding to promoter DNA, thus facilitating the first step in pre-initiation complex formation. This demonstrates that TFIIA is an evolutionary conserved general transcription factor important for activator regulated transcription.
The interaction of TFIIA with the general transcription factor IID (TFIID) has been shown to be rate-limiting step in the transcriptional activation process5. TFIIA binds directly to TBP3,4, the DNA binding subunit of the multiprotein TFIID complex12. TBP associated factors (TAFs) 12-14, which are essential for activated transcription, are also required for an activator-dependent stimulation of the TFIIA-TFIID-promoter complex6. While TFIIA has no known function in unregulated basal transcription15, it has been postulated that TFIIA plays a role in preventing inhibitors of TFIID from repressing transcription16-19.
A TFIIA homolog has been identified in yeast9-10, and the genes encoding the two subunits are essential for viability11. Human TFIIA consists of three polypeptides (xcex1, xcex2, xcex3), but the two largest subunits are derived from a single gene which shares homology to the large subunit of yeast TFIIA7,20,21. Both the human and yeast protein bind to the evolutionary conserved domain of TBP22 and stimulate transcription reconstituted with TFIID, but not TBP17,19.
The polypeptide of the present invention has been putatively identified as the xcex3 subunit of TFIIA. This identification has been made as a result of amino acid sequence homology.
In accordance with one aspect of the present invention, there is provided a novel mature polypeptide which is the small subunit of TFIIA, as well as fragments, analogs and derivatives thereof. The polypeptide of the present invention is of human origin.
In accordance with another aspect of the present invention, there are provided polynucleotides (DNA or RNA) which encode such polypeptide.
In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptide by recombinant techniques.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptide, or polynucleotide encoding such polypeptide for therapeutic purposes, for example, for regulating gene-specific or global transcription and to counteract repressors of the TFIID complex.
In accordance with yet a further aspect of the present invention, there are provided antibodies against such polypeptides.
In accordance with yet another aspect of the present invention, there are provided antagonists to such polypeptides, which may be used to inhibit the action of such polypeptides, for example, to inhibit transcription of undesired cells, e.g., malignancies.
These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.